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Abstract
Glycoproteins are an important class of biomolecule involved in many processes of biomedical interest including cell communication, cancer progression, and pathogen-host interactions. Despite their importance, methods for structural analyses of glycoproteins have lagged behind other classes of proteins. This is largely due to the heterogeneous carbohydrate post-translational modifications typical of glycoproteins. Here, several advancements in methodology for studying glycoproteins with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are presented. For NMR, a new pulse sequence for measurement of residual dipolar couplings in 13C-methyl groups using direct carbon observation has been developed. An updated program for resonance assignments of sparsely labeled proteins, Assign_SLP_GUI, has been developed. This program incorporates additional types of data, pseudocontact shifts (PCS) and paramagnetic relaxation enhancements, which can be obtained from paramagnetic tagging experiments. The sparse labeling assignment strategy, along with a structural interpretation of PCSs was then used to study the interdomain orientation of a glycoprotein construct engineered with a lanthanide binding peptide sequence, hRobo1-Ig1-Ig2-LBP4, both with and without a bound ligand. PCS measurements were used to determine optimal interdomain orientations with and without bound ligand. Lastly, a combination of top-down and bottom-up mass spectrometry was used to measure the glycan occupancy of hCEACAM1-IgV with implications for the function of OST-B, one of the proteins responsible for the initial step of adding an N-glycan to a glycoprotein. Together, these results highlight the complementary nature of NMR and MS for the analysis of glycoproteins.